J Arthropod-Borne Dis, June 2016, 10(2): 211–221 Ch Zhang et al.: Evaluation of Isotope … 211 http://jad.tums.ac.ir Published Online: January 05, 2016 Original Article Evaluation of Isotope 32P Method to Mark Culex pipiens (Diptera: Culicidae) in a Laboratory Chongxing Zhang 1, 2, Guihong Shi 1, Yuqiang Zhao 1, Dongmei Yan 1, Huaiju Li 1, Hongmei Liu 1, *Itsanun Wiwatanaratanabutr 3, *Maoqing Gong 1 1Shandong Institute of Parasitic Diseases, Shandong Academy of Medical Sciences, Jining, Shandong, People’s Republic of China 2Collaborative Innovation Center for the Origin and Control of Emerging Infectious Diseases, Taishan Medical University, Taian, Shandong 271000, China 3Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkut's Insti- tute of Technology Ladkrabang, Bangkok 10520, Thailand (Received 16 Jan 2014; accepted 20 May 2015) Abstract Background: The aim of the current study was to develop a marking technique as an internal marker to mark post blood meal mosquitoes by using stable phosphate isotope 32P and determine the optimal concentration of it. Methods: An isotonic physiological saline solution, containing different concentration of radioactive isotope 32P- labeled disodium phosphate (Na2H 32PO4) was injected into rabbits via the jugular vein in the laboratory. Emerged Cx. pipiens were marked after feeding on rabbit. At the same time, the labeled conditions of emerged Cx. pipiens were also measured by placing feces of No. 6 rabbit into containers with mosquito larvae and pupae inside. Results: According to the label condition of Cx. pipiens after taking blood and the effect of different dosage Na2H 32PO4 on rabbit health, the optimal concentration of radioactive isotope was determined, that is, 0.1211 mCi/kg. By placing feces of No. 6 rabbit into containers with mosquito larvae and pupae inside, the emerged mosquitoes were also labeled. Therefore, feeding mosquitoes on the animal injected with radioactive Na2H 32PO4 was more prac- tical for detecting and tracing mosquitoes. Conclusion: The method was less time-consuming, more sensitive and safer. This marking method will facilitate post-bloodmeal studies of mosquitoes and other blood-sucking insects. Keywords: Radioactive isotope, Mark, Culex pipiens, Rabbit Introduction Mosquitoes, the most important group of nuisance pest insects, due to their diversity and abundance, demonstrated vector competence and frequent infection in nature, they are re- garded as one of the most important vectors of diseases (Sardelis et al. 2002, Molaei et al. 2006, Burkett-Cadena et al. 2008a, b). Track- ing the movement of mosquitoes in their nat- ural habitat is critically important for under- standing their basic biology, demography, ethology and vector-borne disease control as well as prevention. A reliable method for mark- ing is critical important to study mosquito be- havioral characteristics. Animal marking have been used since 218 BC when Fisher and Peterson used banding to distinguish ownership of birds (Fisher and Peterson 1964). Unfortunately, for marking insects, most marking techniques for verte- brate, such as bands, brands, tattoos, tags, notches, paints are not practical because they are tedious, time-consuming, heavy and cost- ly (Southwood 1978, Basavaraju et al. 1998). Insect marking for scientific studies dates back to 1920, since then, a variety of marking techniques, paints, dyes etc. were used to in studies of insect population dynamics (Gei- ger et al. 1919, Dudley and Searles 1923). *Corresponding author: Dr Maoqing Gong, E -mail: maoqingg@yahoo.com, Dr Itsanun Wiwatanaratanabutr, E-mail: kwitsanu@kmitl.ac.th J Arthropod-Borne Dis, June 2016, 10(2): 211–221 Ch Zhang et al.: Evaluation of Isotope … 212 http://jad.tums.ac.ir Published Online: January 05, 2016 Methods to mark mosquitoes have included dyes (Welch et al. 2006, Midega et al. 2007), paints (Trpis and Hausermann 1986, Niebyl- ski and Meek 1989, Service 1993, Bellini et al. 2010, Ciota et al. 2012, Liu et al. 2012, Ver- hulst et al. 2013), dusts (Reisen et al. 1978, Reisen et al. 1992, Russell et al. 2005), trace elements (Anderson et al. 1990, Holbrook et al. 1991, Solberg et al. 1999, Wilkins et al. 2007), and radioactive isotopes (Jenkins 1949, Abdel-Malek 1966, Lindquist et al. 1967, Hood-Nowotny et al. 2006, Hamer et al. 2012). However, for study on the behavioral char- acteristics of post bloodmeal, existing tech- niques tend to be labor intensive, as they re- quire rearing mosquitoes, marking them in large quantities, and then inspecting large numbers of individuals to detect recaptures (Walker et al. 1987). Furthermore, compared with natural populations, rearing mosquitoes, marking them in large quantities by using ar- tificial methods, and releasing them may change their behavior (Reisen et al. 2003, Silver 2008). For study the behavior charac- teristics after blood meal, these methods are not ideal. However, the problem is how to mark breeding mosquitoes without inhibiting their normal biology, and with long-term retention after blood meal, it is still bothering most biological scientists. Until now, only Zhang et al. (2014) marked adult mosquitoes by feeding them on cow injected with iso- tope 32P and subsequent ecological investi- gations. In some studies, successfully labeled mosquitoes by feeding them on radioactive animal blood (Hassett and Jenkins 1951), use of large bait animals for marking wild pop- ulation of adult Anophels aquasalis injected with a dose of 1.7 curies of 32P (Bruce-Chwatt 1956). However, such high dosage would be dangerous to the animal (Winteringham Lon- don meeting, 1953). The aim of the current study was to de- velop a marking technique as an internal marker to mark post blood meal mosquitoes by using stable phosphate isotope 32P and determine the optimal concentration of it. Injection of rabbits with Na2HP 32O4 and blood feeding of Culex pipiens Before experimenting, 6 healthy rabbits (2 kg) were selected and physically exam- ined by veterinarian. The injection method of normal saline solution to the rabbits was according to Smith et al. (1951), i e. An iso- tonic buffered saline solution, containing different concentration of Na2HP 32O4, was injected intravenously of rabbits. The dosage for No. 1, 2 rabbits, No. 3, 4 rabbits, No. 5 rabbit and No. 6 were 0.2 mCi, 0.4 mCi, 0.8 mCi and 1.7 mCi, respectively. For the neg- ative control, 1 healthy rabbits was injected with isotonic buffered saline solution with- out Na2HP 32O4.Then 20 to 50 emerged fe- male Cx. pipiens fed on these rabbits at 6, 12, 24, 48, 72 h and 120 h after injected with Na2HP 32O4. At the same time, 0.2 ml of blood was extracted from rabbits, as well as at 16th and 32nd days for radioactivity level measure. Every batch of Cx. pipiens’ radio- activity levels were measured at 2 h, 6 h, 12 h, 24 h, 48 h, 72 h and 120 h after blood meal. Measuring methods The measurement of radioactivity was con- ducted using a Liquid Scintillation Counters (Model YSJ-76). Before injection of 32P to rabbits, to normalize background radiation, radioactivity level of 30 emerged adult Cx. pipiens reared in the lab was measured. Mos- quitoes tested were anesthesia with ether, placed in a β bell counter tube of the vitriol chambers for 1 minute, as for the rabbits blood test, 0.2 ml of blood was dipped on the paper in the tinfoil sample plates, counts that exceeding 50% of the background was as the standard that were labeled. Placing feces of No. 6 Rabbit in tap water to mark mosquitoes At the 2nd day after No. 6 rabbit was in- J Arthropod-Borne Dis, June 2016, 10(2): 211–221 Ch Zhang et al.: Evaluation of Isotope … 213 http://jad.tums.ac.ir Published Online: January 05, 2016 jected with Na2H 32PO4, up to 5 g feces was placed into a container with 300 ml of tap water and Cx. pipiens’ larvae and pupae in- side. As the negative control, placed 5 g fe- ces of the rabbit without injected Na2HP 32O4 into the water. Radioactivity levels of emerged mosquitoes were measured. Ethics clearance The experimental project was reviewed and approved by the Ethical Committee of Shandong Academy of Medical Sciences (Jinan, Shandong). Urine and feces of the rabbits were collected and sent to the Insti- tute of Radiation Medicine, Shandong Acad- emy of Medical Sciences for appropriate processing to prevent spread of the isotope. The half-life of 32P was 14.3 d. Results Conditions of mosquitoes radioactively la- beled after blood feeding Radioactivity level of emerged adult Cx. pipiens and rabbits blood was measured, the background was determined as was ～12–13 counts per minute (CPM), i e counts that ex- ceeded 50% of background (>20 cpm) were considered positive. Within 5 days after No. 1 to 5 rabbits were injected with Na2H 32PO4, 579 female Cx. pipiens fed on the rabbits, among the 222 mosquitoes blood feeding, except one mosquito was fewer than 20 CPM, the others 221 mosquitoes were labeled no matter how much dosage of Na2H 32PO4 was injected. The more dosage the rabbits injected with Na2H 32PO4, the higher radioactive levels of Cx. pipiens after blood feeding. The radio- active levels started to decrease from the 48 hours (2nd day) after injection. Compared with radioactive level at 48 h, though the radioac- tive level of labeled mosquitoes was higher than background at 72 h, 96h and 120 h, ra- dioactive level decreased to a lower level (Figs. 1, 2, 3). Radioactivity levels of rabbit blood after injection of Na2HP 32O4 The radioactive level decreased very fast in the blood of rabbits. At 6 hour the radio- active level was 100%, the radioactive level decreased 26–48%, 60–75% and 95–97% at the 2nd, the 5th and the 32nd day, respectively (Table 1). Effect of Na2H 32PO4 on rabbit health No. 1 to 4 rabbits showed good health, have no change on body temperature and body weight. On the 2nd day, No. 5 rabbit abortion, and gave birth to 2 died bunnies, the radioactive level was 15 mR / h at 5 cm away from these died bunnies. The No. 5 rabbit presented no abnormalities and was dissected at the 143rd day after injected with Na2H 32PO4, no pathological changes were found in the internal organs. The radioactiv- ity level of liver was measured, which de- creased to similar level of background. However, No. 6 rabbit showed diarrhea, ap- petite loss, weight loss and other symptoms. At the 31st days, and no special lesions were found in internal organs after dissection. It still presented a higher radioactivity level in liver, spleen, intestine, heart, lung, kidney, muscle etc (Table 2). Conditions of mosquitoes radioactively la- beled after placing feces in the container At the 2nd day after No. 6 rabbit was in- jected with Na2H 32PO4. After 5 g feces of No. 6 rabbit was placed into a container, at the 2nd and 7th day, there were total 39 adult Cx. pipiens mosquitoes emerged, among them, 22 (56.5%) were marked, their CPM was 21～875, the average was 323. J Arthropod-Borne Dis, June 2016, 10(2): 211–221 Ch Zhang et al.: Evaluation of Isotope … 214 http://jad.tums.ac.ir Published Online: January 05, 2016 Counts per minute (CPM) of mosquitoes after blood-feeding (hours) 0 20 40 60 80 100 120 2 h 6 h 12 h 24 h 48 h 72 h 96 h 120 h CPM 6 12 24 48 72 120 Fig. 1. Levels of radioactivity of Culex pipiens after fed on No. 1, 2 rabbits after injection of 32P (0.2 mCi) Legend 6, 12 , 24, 48, 72, 120: mosquito fed time after injection 32P into rabbit (hours) Counts per minute (CPM) of mosquitoes after blood-feeding (hours) 0 50 100 150 200 250 2 h 6 h 12 h 24 h 48 h 72 h 96 h 120 h CPM 6 h 12 h 24 h 48 h 72 h 120 h Fig. 2. Levels of radioactivity of Culex pipiens after fed on No. 3, 4 rabbits after injection of 32P (0.4 mCi) Legend 6, 12, 24, 48, 72, 120: mosquito fed time after injection 32P into rabbit (hours) Counts per minute (CPM) of mosquitoes after blood-feeding (hours) 0 100 200 300 400 500 2 h 6 h 12 h 24 h 48 h 72 h 96 h 120 h CPM 6 24 48 72 120 Fig. 3. Levels of radioactivity of Culex pipiens after fed on No. 5 rabbits after injection of 32P (0.8 mCi) Legend 6, 24, 48, 72, 120: mosquito fed time after injection 32P into rabbit (hours) J Arthropod-Borne Dis, June 2016, 10(2): 211–221 Ch Zhang et al.: Evaluation of Isotope … 215 http://jad.tums.ac.ir Published Online: January 05, 2016 Table 1. Radioactivity levels of rabbit blood after injection of 32P (1.7 mCi) at different time Rabbit No. Dosage (mCi) Radioactivity level of rabbit blood after injection of 32P 6h 12h 24h 2 days 3 days 5 days 16 days 32 days C P M C P M D ec re as e% C P M D ec re as e% C P M D ec re as e% C P M D ec re as e% C P M D ec re as e% C P M D ec re as e% C P M D ec re as e% 1, 2 0.2 2709 1922 29 1701 37 1413 48 992 63 681 75 226 92 84 97 3, 4 0.4 4155 3383 19 2926 30 2383 43 1820 56 1199 71 317 92 120 97 5 0.8 5701 5189 9 5290 7 4234 26 3681 35 2300 60 783 86 261 95 6 1.7 9284 - - - - - - - - - - 1,426 85 - - CPM = pulse per minute, - Do not measured Table 2. Radioactivity levels in No. 6 rabbit tissue after injection of 32P (1.7 mCi) 31 days Tissue Weight (mg) CPM CPM per tissue Liver 275 5,263 19,138 Muscle 295 5,586 19,000 Spleen 197 3,118 15,827 Heart 336 5,170 15,387 Small intestine 205 2,419 11,800 Lung 270 2,685 9,944 Kidney 306 2,628 8,588 Background radiation= 22 CPM 215 J Arthropod-Borne Dis, June 2016, 10(2): 211–221 Ch Zhang et al.: Evaluation of Isotope … 216 http://jad.tums.ac.ir Published Online: January 05, 2016 Discussion We could see that the more dosage the rabbits injected with Na2H 32PO4, the higher radioactive levels of Cx. pipiens after blood feeding. The radioactive levels started to de- crease from the 48 hours (2nd day) after in- jection. Compared with radioactive level at 48 h, though the radioactive level of labeled mosquitoes was higher than background at 72 h, 96 h and 120 h, radioactive level de- creased to a lower level. The more injection of Na2H 32PO4 into rabbits, the higher radio- activity level in the blood and the slower de- crease, was also consistent with the labeling condition of Cx. pipiens (Figs. 1, 2, 3). No. 1 to 4 rabbits showed good health, but as for the No. 5 rabbit injected with 0.8 mCi of Na2H 32PO4, on the 2 nd day, abortion, and gave birth to 2 died bunnies. At the 143rd day, the No. 5 rabbit presented no ab- normalities, no pathological changes were found in the internal organs. The radioactivi- ty level of liver was similar to background. However, No. 6 rabbit injected with 1.7 mCi of Na2H 32PO4 showed diarrhea, appetite loss, weight loss and other symptoms. At the 31st days, it still presented a higher radioactivity level in liver, spleen, intestine, heart, lung, kidney, muscle etc. (Table 2). Therefore, the appropriate concentration for not only mark- ing mosquitoes but also no harm to rabbits was not more than 0.4 mCi. Stable isotopes occur naturally in the en- vironment, are safe and non-invasive, pose no health or environmental risks (Hood-Nowotny and Knols 2007). In medical research, most stable isotopes are non-toxic and are routine- ly used for mosquito feeding trials, in which human adults are ‘labelled up’ through sup- plementary feeding with stable isotopes, may be useful for host seeking behavior and re- pellent testing, etc., in ‘real’ environments. Sev- eral tracers were studied, such as 60Co, 89Sr, 65Zn, 131I, 45Ca and 32P, whereas 32P was the most applied radioisotope for tagging due to its short half-life, safety, activity and easy of detection (O’Brien and Wolfe 1964). One of the earliest examples of using inorganic 32P labelled Ae. aegypti mosquitoes was report- ed by Hasset and Jenkins (1949). Hasset and Jenkins (1951) also performed a detailed study of the conditions affecting mosquitoes la- belled with 32P and compared stages, 32P con- centrations and age. The filarial larvae, Setar- ia digitata Linstow was marked after adult mosquitoes (Armigerea obturbans Walker) fed on cows or men infected with microfilaria, that larvae of mosquitoes were reared in wa- ter containing 1 μc of 32P/mL (Dissanaike et al. 1957). Toxicity to the insect was also a serious problem to be considered in many studies (Quarterman et al. 1955). The radioisotopes 45Ca and 131I were very toxic when fed to adult houseflies at 1 μc/mL of milk, whereas 32P was satisfactory (Quarterman et al. 1954). By using 15N-labeled potassium nitrate and 13C-labeled glucose to mark larval mosqui- toes, there were no consistent effects of iso- topic enrichment on immature mosquito sur- vival or adult mosquito body size (Hamer et al. 2012). Although fluorescent dyes or powders are also suitable for marking mosquitoes (Takken et al. 1998, McCall et al. 2001, Pates 2002, Lapointe 2008, Baber et al. 2010, Bellini et al. 2010), and no effect of these dyes and powders on performance of ant (Pogonomyr- mex owyheei), mountain pine beetle (Den- droctonus ponderosae), grasshopper (Mela- noplus spp.), Ae. aegypti, Anopheles sinensis in some studies (Porter and Jorgenson 1980, Linton et al. 1987, McMullen et al. 1988, Narisu et al. 1999, Liu et al. 2012, Valerio et al. 2012). Others have found a reduced lon- gevity, behavioural response or survival of opine parasitoids (Diachasmimorpha spp.), weevils, Ae. aegypti, Asian citrus psyllid (Di- aphorina citri) and codling moths (Laspeyre- J Arthropod-Borne Dis, June 2016, 10(2): 211–221 Ch Zhang et al.: Evaluation of Isotope … 217 http://jad.tums.ac.ir Published Online: January 05, 2016 sia pomonella) (Sheppard et al. 1969, Mof- fitt and Albano 1972, Reinecke 1990, Mess- ing et al. 1993, Nakata 2008, Verhulst et al. 2013). As for the fluorescent marker, be caused of the restrictions of the retention, most stud- ies researches primarily focus on dispersal of nulliparous female mosquitoes during the in- itial host-seeking event and sometimes a se- cond host-seeking event (Hamer et al. 2012). As for trace element, mosquitoes were successfully marked after feeding on hosts injected with trace element Rb and Cs (An- derson et al. 1990, Solberg et al. 1999). Mark- ing with trace elements has many advantages over other insect-marking procedures. They are not radioactive, safe for workers and for the environment. As for insects marked with an element, no tags, paints, dyes, dusts, or visible marks were found left to alter insects behavior or interactions with other insects (Hagler and Jackson 2001). However, a limitation to use of trace el- ements as insect markers in large fields is that the detection of elements can be diffi- cult, expensive, and time-consuming, requires technical expertise and expensive detection equipment (Akey and Burns 1991). Some trace elements are not retained very well in certain insect species, for example, Rb could be detected for only 2–6 days after marking aphids (Guillebeau et al. 1993) and adult Lygus lineolaris (Fleischer et al. 1986). High concentrations of trace elements can adverse- ly affect development, survival, increased adult deformity, reduced pupation, eclosion, egg production and fecundity of certain in- sects (Stimmann et al. 1973, Hayes 1989, Knight et al. 1989, Van Steenwyk et al. 1992). Naturally, occurring stable isotope mark- ers are useful, as they do not require the pre- marking of individuals (Hood-Nowotny and Knols 2007). Labelling a distinct portion of an ecosystem with stable isotopes is a useful, minimally invasive method to study insect dispersal from an ecophysiological perspec- tive (Macneale et al. 2004, 2005). Stable iso- topes occur naturally in the environment, un- like painting, dusting, etc., stable isotope meth- ods are non-invasive and samples require on- ly minimal preparation following collection, which makes the cost of the process as a completely comparable to methods such as polymerase chain reaction (Hood-Nowotny and Knols 2007). Other advantages are the analysis costs (depending on the isotope and the matrix, the cost per sample may range from US$ 5–100.00), shipping stable isotope samples is simple, safe and inexpensive (IAEA 2009). It would cost between $150– 250 to label 1 000 000 Anopheles mosqui- toes with 13C-labelled glucose in the larval stages (Hood-Nowotny et al. 2006). It is possible to trace the fate of labelled sperm into female spermatheca in studies of male mosquitoes labelled with 13C (Helinski et al. 2007). These stable isotope markers meet the usual criteria for use in insect studies: reten- tion, no effect on behavior, durability, easily applied, clearly identifiable, and not expen- sive (Hagler and Jackson 2001). A labelled blood source also provides an easily identifiable point source for post feed- ing dispersal studies. Tracing of labelled blood to determine resource allocation to the eggs or other tissues could also provide useful phys- iological information. It was possible to mark large numbers of mosquitoes by providing blood meals from a host fed injected with a stable isotope substance. Conclusion Na2H 32PO4 was injected via the jugular of rabbit vein; these rabbits were bitten by emerged female Cx. pipiens, so mosquitoes were successfully marked. The appropriate dosage (not only can label mosquito but also have no ill effects on rabbits) of Na2H 32PO4 was 0.1 mCi/kg. The emerged adult Cx. pipiens mosquitoes were also can be marked by placing rabbit feces or Na2H 32PO4 into J Arthropod-Borne Dis, June 2016, 10(2): 211–221 Ch Zhang et al.: Evaluation of Isotope … 218 http://jad.tums.ac.ir Published Online: January 05, 2016 container. The technique was less time-con- suming, more sensitive, and safer, can be used to study post-blood meal dispersal of mosquitoes and other blood-sucking insects. The short half-life of 14 days of 32P, howev- er, was a disadvantage in studies where pro- longed observations were necessary. Acknowledgements We gratefully acknowledge the support and technical assistance provided by Mr Tianbao Fan. The Project Sponsored by Shan- dong Natural Science Foundation (Grant No. ZR2014YL038). The Scientific Research Foun- dation for the Returned Overseas Chinese Scholars, State Education Ministry, Joint Re- search and Development Project Under the twenty first Session of the Sino-Thai Scien- tific and Technical Cooperation (Grant No. 21-RD-05). Shandong Academy of Medical Sciences Foundation (2013–2016). The authors declare that there is no conflict of interests. References Abdel-Malek AA (1966) Study of the dis- persion and flight range of Anopheles sergentii Theo in Siwa Oasis using ra- dioactive isotopes as markers. Bull World Health Organ. 35: 968–973. Akey DH, Burns DW (1991) Analytical con- sideration and methodologies for ele- mental determinations in biological samples. Southwest Entomol. 3: 25–36. 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